#4 - Transcription of RelA-SpoT protein genes during Rickettsia rickettsii infection

Abstract

Rickettsia rickettsii is a tick-borne bacterium which causes Rocky Mountain spotted fever in dogs and humans. Maintenance of rickettsiae through different life stages and generations of ticks and transmission from ticks to animals and humans requires adaptations to changing temperatures and blood content. RelA/SpoT family proteins are regulators of stringent responses in many bacterial systems. The genome of R. rickettsii encodes six proteins annotated as RelA/SpoT homologues. We hypothesis that these SpoT proteins play a role in regulating cellular adaptations in R. rickettsii. The purpose of this project was to determine if six RelA/SpoT proteins are transcribed during R. rickettsii infection of human endothelial cells (HMEC-1) and if medium starvation impacts their regulation. Replicate 12-well plate HMEC-1 cultures were infected with R. rickettsii; some cells were kept in depleted media between 24 and 48 hr followed by addition of fresh complete media between 48-72 hrs. Total RNA was extracted from cells harvested at 0, 3, 24, 48, and 72 hours of infection. cDNA was synthetized by reverse transcription reaction. Quantitative PCR assays targeting rickettsial 16S rRNA and six relA/spoT genes were performed to quantify cDNA for each sample. Changes in gene expression were calculated using the 2^-∆∆Ct method and normalized to the expression of 16S rRNA gene. Transcription of RelA, SpoT1, and SpoT2 genes was most elevated at 24 and 48 hr of infection but declined at 72 hr with death of the cells. Transcription of SpoT4 and SpoT5 genes did not change as much during the same time. Only RelA gene transcription was upregulated in starved cells from 24-48 hr relative to fed cells while SpoT1 and SpoT2 expression was reduced. These observations suggest that some RelA/SpoT family proteins play a role in R. rickettsii responses to environmental conditions. The functions of these proteins in rickettsiae need further investigations.